The carbohydrate antigens GloboH, stage-specific embryonic antigen-3 (SSEA3), and stage-specific embryonic antigen-4 (SSEA4) are closely related to one another in either structure or in function. GloboH, SSEA3 and SSEA4 are globo-series glycosphingolipids, with SSEA3 being the non-fucosylated pentasaccharide precursor structure of GloboH, SSEA4 is sialylated SSEA3 with sialic acid α2-3 links to the non-reducing end of galactose of SSEA3.
Stage-specific embryonic antigen-3 (SSEA3) was first identified and defined by the reactivity of an IgM monoclonal antibody generated in a rat immunized with 4- to 8-cell stage mouse embryos. This monoclonal antibody reacted with all mouse preimplantation embryos from oocytes up to the early blastocyst stage where its expression became more restricted, in the primitive endoderm after implantation. The SSEA3 antigenic determinant was determined to be a carbohydrate present on glycolipids and glycoproteins; it was also found on human teratocarcinoma cells and human erythrocytes. In a panel of structures isolated from the 2102Ep human teratocarcinoma cell line, the SSEA3 antibody had the highest affinity for Gal β (1-3)GalNAcβ (1-3)Gal α (1-4)Gal β (1-4)Glc β (1)Cer. This structure is also known as Gb5, galactosyl-globoside, or globopentaosylceramide.
Synthesis of SSEA3 occurs when β 1,3-galactosyltransferase V (β3GalT-V) transfers galactose to the GalNAc of globoside to form Gb5 or galactosyl-globoside. It was determined that SSEA3 was not expressed in hematopoietic or mesenchymal stem cells. Based on immortalized lymph node lymphocytes from primary lung cancer patients, generated hybridomas, and selected for antibody secreting clones; monoclonal antibodies were then generated from two of these clones—J309 and D579, which recognized the SSEA3 antigenic determinant. The antibodies recognized SSEA3 on several tumor cell lines including lung and breast cancer cell lines, and a teratocarcinoma cell line; in an immune adherence assay, rodent monoclonal SSEA3 antibody, also referred to as MC631, reacted against the same cell lines as the J309 and D579 antibodies. SSEA3 has also been found on testicular germ cell tumors, as well as in breast cancer and in BCSCs (breast cancer stem cells).
Chang et al. looked at SSEA3 expression on normal tissues using a tissue microarray because its location outside of cancer and development was largely unknown. The group found SSEA3 to be expressed on normal epithelium of colon, esophagus, small intestine, kidney, prostate, rectum, skin, testis, thymus, and uterine cervix. Expression was located only on the apical surfaces of epithelial cells or in the cytoplasm, which are considered immune system restricted or inaccessible sites. In an experiment using a KLH conjugated GloboH monovalent vaccine in mice, an antibody response was made to only the GloboH antigen. When α-GalCer was added as an adjuvant, the amount of overall antibody production increased and the mice made polyclonal antibodies to both the GloboH, the SSEA3 and the SSEA4 antigen structures, which vaccination was unable to generate in the absence of the adjuvant. This result showed that SSEA3, GloboH and SSEA4 could make promising targets for cancer vaccines and could be targeted simultaneously.
However, most tumor associated carbohydrate antigens have poor immunogenicity and many approaches have been developed to increase the immune response of carbohydrate-based vaccines, including conjugation with a carrier protein, administration with an immunologic adjuvant using unnatural glycosidic linkage, clustered antigens, unimolecular polyvalent vaccine or hetero-glycan multivalent vaccine. Using these strategies, a few carbohydrate-based vaccines that could elicit significant immune responses to target glycan structures were designed for cancer therapy and entered clinical trials. Among them, the clinical trials of Theratope and GMK with adjuvant QS-21 failed to produce statistically significant difference between time-to-disease and overall survival rate. Mot likely these two vaccines could not elicit robust T cell-dependent immune response in patients. Specifically, Theratope and GMK induced a higher level of IgM in patients but could not induce a strong immune IgG response, which is a major problem in carbohydrate-based vaccine development.
Previous studies showed that modification of carbohydrate antigen structures (MCAS) could effectively elicit a higher level of immune response. For example, in the modification study of the capsular polysaccharide of group B meningococci, the N-acetyl groups of α-(2,8)-linked polysialic acid (PSA) was replaced with the N-propinoyl group and such a modification elicited a high antibody response to recognize not only the N-propinoyl PSA, but also the nature N-acetyl PSA. Similar approaches were applied to STn and GM3 antigens to produce high antibody titers against modified and nature forms. The results indicated that N-phenylacetyl, N-fluoroacetyl or N-difluoroacetyl modifications on glycan antigens could improve the immunogenicity. Moreover, the Schultz group reported that incorporation of a p-nitrophenylalanine into the tumor necrosis factor-α (TNF-α) could break immune tolerance and induce more antibody response to TNF-α. Using glycans as antigens, although some progress has been achieved, most cases are the N-modification of disaccharide (STn), trisaccharide (GM3) and polysialic acid (PSA) and some are based on fluorinated MUC1 glycopeptide antigens.